Search

State-of-the-art immunosuppressive therapies, though efficient in the treatment of rheumatic inflammatory disorders, in most cases do not provide a cure. Complete immunablation and the associated deletion of the reactive immunological memory followed by reconstitution of the "healthy" immune system from stem cells results in a therapy-free remission in many patients suffering from rheumatic diseases. Chronically activated T helper (Th) lymphocytes seem to play a critical role in this reactive pathogenic memory. We have shown that the transcription factor twist1 is specifically expressed in chronically activated, inflammatory Th cells. Twist1 as a biomarker for such Th cells enables the identification and elucidation of the role of Th cells in chronic inflammation and opens new therapeutic options for the targeted manipulation of the pathogenic, reactive memory, such as the targeted deletion of twist1 expressing pathogenic Th cells.

Naive T helper (Th) lymphocytes are induced to express the il4 (interleukin-4) gene by simultaneous signaling through the T cell receptor and the interleukin (IL)-4 receptor. Upon restimulation with antigen, such preactivated Th lymphocytes can reexpress the il4 gene independent of IL-4 receptor signaling. This memory for expression of the il4 gene depends on epigenetic modification of the il4 gene locus and an increased expression of GATA-3, the key transcription factor for Th2 differentiation. Here, we have identified a phylogenetically conserved sequence, the conserved intronic regulatory element, in the first intron of the il4 gene containing a tandem GATA-3 binding site. We show that GATA-3 binds to this sequence in a position- and orientation-dependent manner, in vitro and in vivo. DNA demethylation and histone acetylation of this region occurs early and selectively in differentiating, IL-4-secreting Th2 lymphocytes. Deletion of the conserved element by replacement of the first exon and part of the first intron of the il4 gene with gfp leads to a defect in the establishment of memory for expression of IL-4, in that reexpression of IL-4 still requires costimulation by exogenous IL-4. The conserved intronic regulatory element thus links the initial epigenetic modification of the il4 gene to GATA-3 and serves as a genetic control element for memory expression of IL-4.

Regulatory T cells (Tregs) fulfill a central role in immune regulation. We reported previously that the integrin alphaEbeta7 discriminates distinct subsets of murine CD4+ regulatory T cells. Use of this marker has now helped to unravel a fundamental dichotomy among regulatory T cells. alphaE-CD25+ cells expressed L-selectin and CCR7, enabling recirculation through lymphoid tissues. In contrast, alphaE -positive subsets (CD25+ and CD25-) displayed an effector/memory phenotype expressing high levels of E/P-selectin-binding ligands, multiple adhesion molecules as well as receptors for inflammatory chemokines, allowing efficient migration into inflamed sites. Accordingly, alphaE -expressing cells were found to be the most potent suppressors of inflammatory processes in disease models such as antigen-induced arthritis.

The delineation of the in vivo role of GATA-3 in human T cell differentiation is a critical step in the understanding of molecular mechanisms directing human immune responses. We examined T cell differentiation and T cell–mediated effector functions in individuals lacking one functional GATA-3 allele. CD4 T cells from GATA-3+/− individuals expressed significantly reduced levels of GATA-3, associated with markedly decreased T helper cell (Th)2 frequencies in vivo and in vitro. Moreover, Th2 cell–mediated effector functions, as assessed by serum levels of Th2-dependent immunoglobulins (Igs; IgG4, IgE), were dramatically decreased, whereas the Th1-dependent IgG1 was elevated compared with GATA-3+/+ controls. Concordant with these data, silencing of GATA-3 in GATA-3+/+ CD4 T cells with small interfering RNA significantly reduced Th2 cell differentiation. Moreover, GATA-3 mRNA levels increased under Th2-inducing conditions and decreased under Th1-inducing conditions. Taken together, the data strongly suggest that GATA-3 is an important transcription factor in regulating human Th2 cell differentiation in vivo.

Th1 cells are prominent in inflamed tissue, survive conventional immunosuppression, and are believed to play a pivotal role in driving chronic inflammation. Here, we identify homeobox only protein (Hopx) as a critical and selective regulator of the survival of Th1 effector/memory cells, both in vitro and in vivo. Expression of Hopx is induced by T-bet and increases upon repeated antigenic restimulation of Th1 cells. Accordingly, the expression of Hopx is low in peripheral, naive Th cells, but highly up-regulated in terminally differentiated effector/memory Th1 cells of healthy human donors. In murine Th1 cells, Hopx regulates the expression of genes involved in regulation of apoptosis and survival and makes them refractory to Fas-induced apoptosis. In vivo, adoptively transferred Hopx-deficient murine Th1 cells do not persist. Consequently, they cannot induce chronic inflammation in murine models of transfer-induced colitis and arthritis, demonstrating a key role of Hopx for Th1-mediated immunopathology.

1,25-dihydroxyvitamin D(3) (calcitriol) regulates immune responses, e.g., inhibits expression of IgE by B cells and enhances expression of IL-10 by dendritic cells and T cells. We report here that activation of human B cells by B cell receptor, CD40 and IL-4 signals induces expression of the gene for 25-hydroxyvitamin-D3-1alpha-hydroxylase (CYP1alpha). Accordingly, these B cells generate and secrete significant amounts of calcitriol. In activated B cells calcitriol induces expression of the genes Cyp24, encoding a vitamin D hydroxylase, and Trpv6, encoding a calcium selective channel protein. Calcitriol enhances IL-10 expression of activated B cells more than threefold, both by recruiting the vitamin D receptor to the promoter of Il-10, and to lesser extent by modulation of calcium-dependent signaling. The molecular link in activated B cells between vitamin D signaling, expression of IgE and IL-10, and their ability to produce calcitriol from its precursor, suggest that pro-vitamin D (25-hydroxyvitamin D(3)) can be used as a modulator of allergic immune responses.

In Th1 and Th2 memory lymphocytes, the genes for the cytokines interleukin (IL)-4 and interferon-gamma (IFN-gamma) are imprinted for expression upon restimulation. This cytokine memory is based on expression of the transcription factors T-bet for IFN-gamma, and GATA-3 for IL-4, and epigenetic modification of the cytokine genes. In Th2 cells, expression of the cytokine IL-10 is also induced by GATA-3. Here, we show that this induction is initially not accompanied by epigenetic modification of the IL-10 gene. Only after repeated restimulation of a memory Th2 cell in the presence of IL-4, extensive histone acetylation of the IL-10 gene is detectable. This epigenetic imprinting correlates with the development of a memory for IL-10 in repeatedly restimulated Th2 cells. In Th1 cells, IL-10 expression is induced by IL-12, but the IL-10 gene lacks detectable histone acetylation. Accordingly, IL-10 expression in restimulated memory Th1 cells remains conditional on the presence of IL-12. This finding defines a potential anti-inflammatory role for IL-12 in Th1 recall responses. While in primary Th1 responses IL-12 is required to induce expression of the pro-inflammatory cytokine IFN-gamma, in secondary Th1 responses IFN-gamma re-expression is independent of IL-12, which still is able to induce expression of the anti-inflammatory cytokine IL-10.

Current therapeutic options that are based on immunosuppression do not provide a cure for the treatment of chronic inflammation. Though more efficient immunosuppression and the introduction of biologicals such as antibodies targeting cytokines have improved clinical outcomes, immunosuppressive therapy has to be continued to be efficient, thus enhancing the risk of adverse events and undesired side effects. Why can immunosuppression ameliorate, even stop, but not cure chronic inflammation? Is chronic inflammation perpetuated beyond suppression by mechanisms independent of the immune system, or is it perpetuated by components of the immune system which are resistant to a block of ongoing immune reactions? One such component of the immune system is immunological memory. This article will review the role of immunological memory in chronic inflammation, as far as we understand it today, and discuss implications for the development of novel therapeutic strategies aiming at a cure for diseases involving chronic inflammation.

A major problem of antibody-based targeting of solid tumors is the poor penetration of antibodies into tumor tissue. Vasoactive immunoconjugates have been proposed as a means of increasing antibody uptake in tumors. In principle, VEGF (also known as vascular permeability factor) could selectively alter vascular permeability, leading to improved tumor targeting. A possible role for VEGF in the targeting of tumor neovasculature has been postulated, based on the overexpression of VEGF receptors in tumor endothelial cells. However, quantitative biodistribution studies on this topic are not available. In this report, we describe the cloning, expression, characterization and biodistribution in tumor-bearing mice of antibodies fused to either VEGF120 or VEGF164 The MAb fragments chosen for analysis were scFv(L19), specific for the ED-B domain of fibronectin, a marker of angiogenesis, and scFv(HyHEL-10), a negative control antibody of irrelevant specificity in mice. Neither unconjugated VEGF nor scFv(HyHEL-10)–VEGF fusion proteins showed accumulation in the tumor (tumor:blood ratios approx. 1 at 4 hr and 24 hr postinjection). By contrast, scFv(L19)–VEGF120 but not scFv(L19)–VEGF164 showed significant accumulation in tumors (tumor:blood ratio = 9.3 at 24 hr) but was not superior to unconjugated scFv(L19). Preinjection of unlabeled scFv(L19)–VEGF120 prior to administration of radiolabeled fusion protein led to increased accumulation of radiolabeled scFv(L19)–VEGF120 in the tumor but only at very high concentrations (20 μg/mouse). © 2002 Wiley-Liss, Inc.

Human monoclonal antibodies are promising agents for the development of more selective anticancer therapeutics. However, the tumor-targeting efficiency of most anticancer antibodies is severely limited by their poor penetration into the tumor mass. Recent studies have shown that a peptide derived from the HIV TAT protein could improve the distribution of cytoplasmic reporter proteins when administered systemically as fusion proteins or cross-linked chimeras. In this article, we tested by quantitative biodistribtution analysis whether conjugation to TAT peptides could improve the tumor targeting properties of scFv(L19)-Cys: an engineered human antibody fragment specific for the ED-B domain of fibronectin, a marker located in the modified extracellular matrix surrounding tumor neovasculature. Our results show that TAT peptides, consisting either of L-amino acids or D-amino acids, can efficiently transduce target cells when conjugated to fluorophores and/or antibody fragments, suggesting a receptor-independent cell entry mechanism. However, conjugation of scFv(L19)-Cys to TAT peptides resulted in a severely reduced tumor targeting performance compared to the unconjugated antibody, as measured in murine F9 teratocarcinoma-bearing mice, after intravenous injection of the radiolabeled antibody preparations. Our results outline the usefulness of TAT peptides for the efficient in vitro transduction of cells with globular proteins. In particular, the use of TAT peptides composed of D-amino acids may significantly reduce proteolytic degradation. At the same time, the poor biodistribution properties of antibody-TAT conjugates cast doubts over the applicability of this methodology for the delivery of biopharmaceuticals in vivo.

Th lymphocytes are considered to be key players in chronic inflammation. Upon repeated re-stimulation and differential instruction, they can develop a stable memory for the expression of distinct genes coding for cytokines, chemokines, adhesion molecules, and so on. This functional memory contributes considerably to the chronicity of inflammation and its eventual refraction to therapy. Here, we describe the global gene expression profiles of in vitro repeatedly reactivated murine Th1 and Th2 cells, and compare them with those of naive and once activated Th1 and Th2 cells, both before and after re-stimulation (Fig. ​(Fig.11). Figure 1 Hierarchical clustering of differentially expressed genes among doublets of the nine investigated cell populations (w, weeks in culture; res, 3 hours re-stimulated) using Affymetrix Mouse Genome 430A 2.0 Arrays and DNA-Chip Analyzer (dChip). Among differentially expressed genes is a novel transcription factor, which down-modulates NF-κB signalling and is exclusively expressed in repeatedly re-activated Th1 cells. High expression of this gene can be detected in Th cells isolated from the site of inflammation of patients suffering from distinct forms of rheumatic diseases or ulcerative colitis, but not in Th cells isolated from healthy tissue or from blood. Expression of this gene in Th cells at the site of inflammation highlights the role of chronic T-cell activation in chronic inflammation and may represent an endogenous mechanism to limit inflammation. Apart from its diagnostic value, this molecular pathway also points to a new therapeutic target.